Enhancement of Anti-Inflammatory Activity of Optimized Niosomal Colchicine Loaded into Jojoba Oil-Based Emulgel Using Response Surface Methodology

Recent progression in investigational studies aiming to integrate natural products and plant oils in developing new dosage forms that would provide optimal therapeutic effect. Therefore, the aim of the present exploration was to inspect the influence of jojoba oil in boosting the anti-inflammatory effect of colchicine natural product. To our knowledge, there is no formulation comprising colchicine and jojoba oil together to form a niosomal emulgel preparation anticipated for topical application. Colchicine is a natural product extracted from Colchicum autumnale that has been evidenced to show respectable anti-inflammatory activity. Owing to its drawbacks and low therapeutic index, it was preferable to be formulated into topical dosage form. The current study inspected colchicine transdermal delivery by developing niosomal preparation as a potential nanocarrier included into emulgel prepared with jojoba oil. Box Behnken design was constructed to develop 17 niosomal emulgel formulations. The optimized colchicine niosomal emulgel was evaluated for its physical characteristics and in vitro release studies. The in vivo anti-inflammatory activity was estimated via carrageenan-induced rat hind paw edema method. The developed colchicine niosomal preparation revealed particle size of 220.7 nm with PDI value 0.22, entrapment efficiency 65.3%. The formulation was found to be stable showing no significant difference in particle size and entrapment efficiency up on storage at 4 °C and 25 °C for 3 months. The optimized colchicine niosomal emulgel exhibited a pH value 6.73, viscosity 4598 cP, and spreadability 38.3 mm. In vitro release study of colchicine from niosomal emulgel formulation was around 52.4% over 6 h. Apparently, the proficient anti-inflammatory activity of colchicine niosomal emulgel was confirmed via carrageenan-induced rat hind paw edema test. Overall, the results recommend the combination of niosomal preparation with jojoba oil-based emulgel that might signify a favorable delivery of anti-inflammatory drug such as colchicine.


Introduction
Utilizing nanotechnology in the field of drug delivery is an advanced way to deliver drugs on a nanoscale to specific organs in a controlled manner to maximize their therapeutic action and reduce the side effects [1]. Various nanocarriers have been developed for entrapping several drugs and intended to be delivered to specific tissues such as nanoemulsion, nanoparticles, liposome, niosome, and others [2]. Niosomes are regarded as

Results and Discussion
Based on our preliminary studies, a dispersion of niosome was successfully developed with an optimum molar ratio of nonionic surfactant and cholesterol 1:1 in order to attain a physically stable niosome.

Entrapment Efficiency
Entrapment efficiency of developed colchicine loaded niosome was successfully assessed using centrifugation method. The higher solubility of colchicine in water and in surfactant/cholesterol lipid mixture provides a high entrapment efficiency of colchicine, that being 65.3 ± 2.2%. Actually, the type of non-ionic surfactant plays a vital role in the percentage of drug that could be entrapped. Span 60 possesses high transition temperature (53 • C), which participates in higher entrapment efficiency [28]. Moreover, collaboration of cholesterol and non-ionic surfactant stimulates the increase in thickness and surface area of the niosomal layers in addition to the rigidization caused by the surfactant, which results in high entrapment [29]. Further, the molar ratio of cholesterol to non-ionic surfactant participates in achieving higher entrapment into the niosomes [30]. Previous literature stated that colchicine was highly entrapped into niosomes upon adjusting the molar ratio to be 1:1 between surfactant and cholesterol [31].

Vesicular Size and Size Distribution (PDI)
Particle size and PDI measurement of the developed colchicine-loaded niosome were estimated and data displayed in Figure 1. It exhibited particle size of 220.7 ± 2.3 nm, with good distribution (PDI 0. 22), which indicate that the particles fall within a narrow range of sizes.

Results and Discussion
Based on our preliminary studies, a dispersion of niosome was successfully developed with an optimum molar ratio of nonionic surfactant and cholesterol 1:1 in order to attain a physically stable niosome.

Entrapment Efficiency
Entrapment efficiency of developed colchicine loaded niosome was successfully assessed using centrifugation method. The higher solubility of colchicine in water and in surfactant/cholesterol lipid mixture provides a high entrapment efficiency of colchicine, that being 65.3 ± 2.2%. Actually, the type of non-ionic surfactant plays a vital role in the percentage of drug that could be entrapped. Span 60 possesses high transition temperature (53 °C), which participates in higher entrapment efficiency [28]. Moreover, collaboration of cholesterol and non-ionic surfactant stimulates the increase in thickness and surface area of the niosomal layers in addition to the rigidization caused by the surfactant, which results in high entrapment [29]. Further, the molar ratio of cholesterol to non-ionic surfactant participates in achieving higher entrapment into the niosomes [30]. Previous literature stated that colchicine was highly entrapped into niosomes upon adjusting the molar ratio to be 1:1 between surfactant and cholesterol [31].

Vesicular Size and Size Distribution (PDI)
Particle size and PDI measurement of the developed colchicine-loaded niosome were estimated and data displayed in Figure 1. It exhibited particle size of 220.7 ± 2.3 nm, with good distribution (PDI 0. 22), which indicate that the particles fall within a narrow range of sizes.

Stability Study
Examining the stability of the developed colchicine-loaded niosome was accomplished and the results are depicted in Figure 2A, B. Non-significant variations were distinguished in the entrapment efficiency and vesicular size of the formulation following 1 and 3 months storage at 4 °C and 25 °C if compared to freshly prepared niosomal preparation (p < 0.05). The study outcomes emphasized the formulation stability and support niosome for a potent drug carrier. Actually, presence of DSPE-PEG represents a key role in increasing formulation stability, since it offers certain steric hindrances for the formulation membrane [32,33].

Stability Study
Examining the stability of the developed colchicine-loaded niosome was accomplished and the results are depicted in Figure 2A,B. Non-significant variations were distinguished in the entrapment efficiency and vesicular size of the formulation following 1 and 3 months storage at 4 • C and 25 • C if compared to freshly prepared niosomal preparation (p < 0.05). The study outcomes emphasized the formulation stability and support niosome for a potent drug carrier. Actually, presence of DSPE-PEG represents a key role in increasing formulation stability, since it offers certain steric hindrances for the formulation membrane [32,33].

Statistical Analysis of the Design
Analysis of variance for all the dependent variables was carried out using BBD software and certain parameters including p-value, F-value, and model F-value were obtained using ANOVA. Primarily, the best model to fit for all responses was the quad-

Statistical Analysis of the Design
Analysis of variance for all the dependent variables was carried out using BBD software and certain parameters including p-value, F-value, and model F-value were obtained using ANOVA. Primarily, the best model to fit for all responses was the quadratic one if compared to all other models. Referring to Table 2, it was noticed that p-value for all responses were less than 0.0001, which is necessary for confirming that the independent variables showed a significant effect on the investigated response. Relating to F-value of the responses, it was proven to be greater values which are recommended to provide less error in the model. The model F-value for R 1 and R 2 is 67.64 and 35.53, respectively, which implies that the model is significant. Regarding lack of fit, it is mandatory to be non-significant in order to fit the model and confirm its efficiency [34]. It was apparent that lack of fit was 2.18 and 2.20, which is not significant relative to the pure error in addition to their related p-value 0.2333 and 0.2304 for R 1 and R 2, respectively, which indicates that the chance for this large value due to noise was 23.33 and 23.04%, respectively.

Effect of Independent Variables on Viscosity (R 1 )
Viscosity is a very important parameter for topical preparations, since it may alter the diffusion of the drug and consequently affect its in vitro release [35]. The viscosity of various formulated colchicine niosomal emulgel was measured and data are summarized in Table 1. The formulations viscosity was extended between 4265 ± 184 and 9834 ± 210 cP. It was highly obvious that a parallel relation was detected between the viscosity of all niosomal emulgel preparations and the concentration of the different independent variables used. Accordingly, upon increasing X 1 , X 2 , and X 3 , a consistent increase in formulations viscosity was observed and this is certainly due to the formulation ingredients [36]. In consequence, greater oil concentration in the formulation contributed to an increment in viscosity. As well, higher concentration of gelling agent gave rise to corresponding increase in formulation viscosity [10]. Regarding the influence of X 2 on the R 1 response, it was revealed that upon using the same concentration of oil and gelling agent, an increase in niosomal emulgel was confirmed. This noticeable impact of the three factors on the viscosity was further illustrated by the following mathematical equation: R1 = 6229.6 + 2124.12 X 1 + 181.125 X 2 + 673.5 X 3 + 264.25 X 1 X 2 + 319 X 1 X 3 + 160 X 2 X 3 + 585.325 X 1 2 − 6.675 X 2 2 − 84.425 X 3 As noted, the positive sign in the equation confirmed the synergistic effect of X 1 , X 2 , and X 3 on R 1 response. As well, the previous positive effect was emphasized and represented by 2D contour graph and 3D-response surface plot as portrayed in Figures 3 and 4. As noted, the positive sign in the equation confirmed the synergistic effect of X1, X2, and X3 on R1 response. As well, the previous positive effect was emphasized and represented by 2D contour graph and 3D-response surface plot as portrayed in Figures 3 and 4.  Furthermore, there was a linear correlation detected between the predicted versus the actual responses where the predicted R 2 for response R1 is (0.8805), which is in reasonable agreement with the adjusted R 2 (0.9740) as illustrated in Table 2. Additionally, Figure 5 presented the residual values that were distributed among the two sides of the line signifying that the actual data and the predicted values were in a credible correlation with each other. Likewise, R 2 value (0.9886) provides an indication that the system could recommend the model, adding to that the adequate precision (27.6989), which is a desirable value elucidating an adequate signal and concluding that the model could navigate the design space.  As noted, the positive sign in the equation confirmed the synergistic effect of X1, X2, and X3 on R1 response. As well, the previous positive effect was emphasized and represented by 2D contour graph and 3D-response surface plot as portrayed in Figures 3 and 4.  Furthermore, there was a linear correlation detected between the predicted versus the actual responses where the predicted R 2 for response R1 is (0.8805), which is in reasonable agreement with the adjusted R 2 (0.9740) as illustrated in Table 2. Additionally, Figure 5 presented the residual values that were distributed among the two sides of the line signifying that the actual data and the predicted values were in a credible correlation with each other. Likewise, R 2 value (0.9886) provides an indication that the system could recommend the model, adding to that the adequate precision (27.6989), which is a desirable value elucidating an adequate signal and concluding that the model could navigate the design space. Furthermore, there was a linear correlation detected between the predicted versus the actual responses where the predicted R 2 for response R 1 is (0.8805), which is in reasonable agreement with the adjusted R 2 (0.9740) as illustrated in Table 2. Additionally, Figure 5 presented the residual values that were distributed among the two sides of the line signifying that the actual data and the predicted values were in a credible correlation with each other. Likewise, R 2 value (0.9886) provides an indication that the system could recommend the model, adding to that the adequate precision (27.6989), which is a desirable value elucidating an adequate signal and concluding that the model could navigate the design space.

Effect of Independent Variables on In Vitro Release Study (R2)
The features of the in vitro release study of colchicine from the prepared niosomal emulgel formulations were competently assessed and the outcome was outlined in Figure 6. The investigation was prolonged for 6 h to achieve percentages of drug release that ranged from 21.5 ± 2.7 to 55.1 ± 3.6. It was remarkable that the percentage of colchicine released from all formulations was affected greatly by the examined independent variables. Generally, increasing oil concentration would cause retardation in the process of drug release from the formulation [37]. Additionally, a decrease in the drug release usually perceived upon increasing the concentration of the gelling agent [38]. In the current study, a considerable decrease in the in vitro release of colchicine from the developed niosomal emulgel formulations was observed while increasing the concentration of all independent factors X1, X2, and X3. The variations in the drug release percentage could be correlated to the formulation viscosity, since higher viscosity can produce resistance to drug dispersion and movement and slow the rate of drug dissolution [39]. In view of that, niosomal emulgel formed of higher concentrations of oil, surfactant, and gelling agent exhibited higher viscosity and consequently showed minor percentage of drug release [36]. The next regression equation interprets the previously stated influence of the independent variables X1, X2, and X3 on the in vitro release response R2: besides, the interactions between the independent variables and the examined response (R2) was further clarified by the model graphs as illustrated in Figure 7 showing 2D contour graph and Figure 8 displaying 3D surface plot

Effect of Independent Variables on In Vitro Release Study (R 2 )
The features of the in vitro release study of colchicine from the prepared niosomal emulgel formulations were competently assessed and the outcome was outlined in Figure 6. The investigation was prolonged for 6 h to achieve percentages of drug release that ranged from 21.5 ± 2.7 to 55.1 ± 3.6. It was remarkable that the percentage of colchicine released from all formulations was affected greatly by the examined independent variables. Generally, increasing oil concentration would cause retardation in the process of drug release from the formulation [37]. Additionally, a decrease in the drug release usually perceived upon increasing the concentration of the gelling agent [38]. In the current study, a considerable decrease in the in vitro release of colchicine from the developed niosomal emulgel formulations was observed while increasing the concentration of all independent factors X 1 , X 2 , and X 3 . The variations in the drug release percentage could be correlated to the formulation viscosity, since higher viscosity can produce resistance to drug dispersion and movement and slow the rate of drug dissolution [39]. In view of that, niosomal emulgel formed of higher concentrations of oil, surfactant, and gelling agent exhibited higher viscosity and consequently showed minor percentage of drug release [36]. The next regression equation interprets the previously stated influence of the independent variables X 1 , X 2 , and X 3 on the in vitro release response R 2 :       On the other track, and as revealed from Figure 9, there was a linear relationship between the predicted and the actual responses, since the predicted R 2 (0.7739) was found to be in a sensible harmony with the adjusted one (0.9510) because the difference is less than 0.2 as demonstrated in Table 2. As well, the value of R 2 (0.9786) indicates that the system could support the model and the adequate precision value (20.4175), which is a recommended value proving that the model could navigate the design space. On the other track, and as revealed from Figure 9, there was a linear r between the predicted and the actual responses, since the predicted R 2 (0.7739) to be in a sensible harmony with the adjusted one (0.9510) because the differ than 0.2 as demonstrated in Table 2. As well, the value of R 2 (0.9786) indica system could support the model and the adequate precision value (20.4175), recommended value proving that the model could navigate the design space.

Optimizing the Investigated Variables
In order to provide the optimal characteristics and appropriate levels of that lead to the utmost value of desirability, an optimization technique was ex A numerical optimization was conducted by the design software next to c various graphs that were produced to illustrate the interaction between the in variables. The optimization process depends on pointing the responses tow criteria that are expected to offer the optimized formula. The selected criteria i were to minimize the vesicular size and maximize the in vitro release. Taki

Optimizing the Investigated Variables
In order to provide the optimal characteristics and appropriate levels of constraints that lead to the utmost value of desirability, an optimization technique was executed [40]. A numerical optimization was conducted by the design software next to constructing various graphs that were produced to illustrate the interaction between the independent variables. The optimization process depends on pointing the responses toward certain criteria that are expected to offer the optimized formula. The selected criteria in our study were to minimize the vesicular size and maximize the in vitro release. Taking into account the post analysis data that were provided in the point prediction, the expected actual values of independent variables were (1.04 g) jojoba oil, (0.9 g) Tween 80, and (0.803 g) Na alginate.
The anticipated values for the responses based on higher desirability were 4187.4 cP for R 1 and 55.53% for R 2 . With regard to the previous predictions, the optimized niosomal emulgel formulation was developed and its obtained responses were matched with the predicted values and as per the data in Table 3, both values were prominently close to each other.

Characterization of Optimized Topical Formulations
The optimized colchicine niosomal emulgel was studied for its characteristics and compared to the conventional colchicine niosomal gel and data are presented in Table 4. Both formulations were homogeneous and showed acceptable physical appearance. The pH is in an appropriate range that insures no skin sensitivity or irritation. For the formulations viscosity, it was assessed and showed that incorporating jojoba oil in niosomal emulgel would result in higher viscosity than niosomal gel; however, viscosity of both preparations being in a good range that could be easily applied topically. As for spreadability, it was measured and their values confirmed that the formulations could spread evenly over the skin. Comprehensively, despite the significant difference that was observed between the formulations in certain parameters, nevertheless, the estimated features of niosomal gel and emulgel incorporating colchicine appear to be satisfactory and appropriate for skin application. Values are stated as mean ± (SD) using student t-test. * p < 0.05 compared to colchicine niosomal gel.

Assessment of In Vitro Drug Release Studies
The percentage of colchicine released from different developed formulations and from colchicine solution across cellophane membranes was performed via Franz diffusion apparatus and results are shown in Figure 10. Almost 98.7% of colchicine was released from colchicine solution within one hour due to higher solubility of the drug. Conversely, it was noticed that 52.4 ± 3.0, 72.67 ± 4.5, and 86.67 ± 4.04% of colchicine was released from colchicine niosomal emulgel, colchicine niosomal gel, and colchicine niosome after 6 h, respectively. The significant reduction (p < 0.05) in the drug release from all formulations under investigation compared to colchicine solution could be ascribed to the well-known fact that niosomal formulation has the ability to form cement layer in the spaces of the niosomal bilayer membrane so it will affect the drug diffusion and slow down its release [41]. Moreover, the viscosity of the preparations plays a crucial role in its in vitro behavior, whereas, certain ingredients like gelling agents provide higher viscosity for the formulation that would support drug retention and allow the release in a controlled pattern. The previous illustration could explain the significant reduce (p < 0.05) of colchicine release from niosomal gel and emulgel formulation compared to colchicine niosome itself, since the former formulations comprise gelling agent. On the other side, the significant difference (p < 0.05) in colchicine release from niosomal emulgel compared with niosomal gel is due to the incorporation of jojoba oil and tween 80 that provides further retention for drug release from emulgel formulation [39]. Results are expressed as the mean ± SD of three experiments. * p < 0.05 compared to colchicine solution; $ p < 0.05 compared to colchicine niosome and # p < 0.05 compared to colchicine niosomal gel.

Release Kinetic Mechanism
The kinetic analysis of all formulations under investigation was studied to show the mechanism by which the drug is released through the cellophane membranes and results are exhibited in Table 5 and Figure 11. Plotting the amount of drug released versus time elucidated that the release of colchicine from all formulations followed Higuchi model as it exhibited the most linear plot and established the greatest value of (r2). Higuchi model confirmed the drug has been released from matrix type with perfect sink conditions that were always achieved in the release environment [42]. Interestingly, the release of water-soluble drugs from several semi-solid formulations is extensively represented by Higuchi model [43].

Release Kinetic Mechanism
The kinetic analysis of all formulations under investigation was studied to show the mechanism by which the drug is released through the cellophane membranes and results are exhibited in Table 5 and Figure 11. Plotting the amount of drug released versus time elucidated that the release of colchicine from all formulations followed Higuchi model as it exhibited the most linear plot and established the greatest value of (r2). Higuchi model confirmed the drug has been released from matrix type with perfect sink conditions that were always achieved in the release environment [42]. Interestingly, the release of water-soluble drugs from several semi-solid formulations is extensively represented by Higuchi model [43].   Figure 12 displays the percentage of swelling following topical application of colchicine niosomal gel and emulgel on carrageenan-induced rat hind paw edema compared to non-treated animal (control). The anti-inflammatory effect was calculated as percentage of swelling over time. The non-treated group depicted the utmost swelling after induction of inflammation after 4 h (101.6%). Likewise, the group treated with the placebo I formulation reached the maximum swelling following 3 h of starting the investigation achieving 92.0% and exhibiting a non-significant difference if compared to control-treated group (p < 0.05). On the other side, a significant reduction of swelling was obtained in placebo II, colchicine orally, colchicine niosomal gel and colchicine niosomal emulgel-treated groups if compared to control group (p < 0.05) where it exhibited 56.4, 48.6, 38.6, and 22.8% swelling, respectively, following 12 h. Incredibly, insignificant difference was observed between placebo II and colchicine orally treated group (p < 0.05), which proves the role of emulgel and mainly, jojoba oil in ameliorating the anti-inflammatory influence. In fact, the influence of jojoba oil in alleviating inflammation in different experimental models was previously established [44]. Prominently, animals treated with colchicine niosomal emulgel showed the most plentiful reduction in percentage of swelling, which is statistically significant (p < 0.05) comparing to all other groups treated with other applied preparations. Subsequently, colchicine niosomal emulgel provides the greatest anti-inflammatory potential. The present investigation offers strong confirmation about the improved permeation and enhanced effect of topical formulation incorporating jojoba oil and colchicine in niosomal emulgel and proposing a synergistic effect between them. The prospective of jojoba oil in lowering inflammation was confirmed earlier in our study that exhibited the synergism between jojoba oil and brucine as anti-inflammatory when incorporated into liposomal emulgel formulation [45].  Figure 12 displays the percentage of swelling following topical application of colchicine niosomal gel and emulgel on carrageenan-induced rat hind paw edema compared to nontreated animal (control). The anti-inflammatory effect was calculated as percentage of swelling over time. The non-treated group depicted the utmost swelling after induction of inflammation after 4 h (101.6%). Likewise, the group treated with the placebo I formulation reached the maximum swelling following 3 h of starting the investigation achieving 92.0% and exhibiting a non-significant difference if compared to control-treated group (p < 0.05). On the other side, a significant reduction of swelling was obtained in placebo II, colchicine orally, colchicine niosomal gel and colchicine niosomal emulgel-treated groups if compared to control group (p < 0.05) where it exhibited 56.4, 48.6, 38.6, and 22.8% swelling, respectively, following 12 h. Incredibly, insignificant difference was observed between placebo II and colchicine orally treated group (p < 0.05), which proves the role of emulgel and mainly, jojoba oil in ameliorating the anti-inflammatory influence. In fact, the influence of jojoba oil in alleviating inflammation in different experimental models was previously established [44]. Prominently, animals treated with colchicine niosomal emulgel showed the most plentiful reduction in percentage of swelling, which is statistically significant (p < 0.05) comparing to all other groups treated with other applied preparations. Subsequently, colchicine niosomal emulgel provides the greatest anti-inflammatory potential. The present investigation offers strong confirmation about the improved permeation and enhanced effect of topical formulation incorporating jojoba oil and colchicine in niosomal emulgel and proposing a synergistic effect between them. The prospective of jojoba oil in lowering inflammation was confirmed earlier in our study that exhibited the synergism between jojoba oil and brucine as anti-inflammatory when incorporated into liposomal emulgel formulation [45].

Conclusions
Colchicine was successfully involved in niosomal preparation that is regarded as a promising drug delivery technique. For better application, the niosomal formulation was incorporated into various emulgel dosage forms. Box Behnken design approach was executed and came up with several niosomal emulgel formulas. The optimized of formula was selected based on values of certain investigated factors that was (1.04 g) jojoba oil, (0.9 g) Tween 80, and (0.803 g) Na alginate, and their obtained responses. The optimized formulation exhibited suitable physical properties including pH 6.73, viscosity 4598 cP, and spreadability 38.3 mm, in addition to an effective in vitro drug release study that reached 52.4% over 6 h. The developed formulations could mitigate hind paw inflammation induced by carrageenan since niosomal emulgel formulation containing jojoba oil could reduce paw swelling to reach 22.8%. In essence, the study revealed that the pharmacological action of colchicine was synergistically enhanced upon merging colchicine niosome with emulgel prepared with jojoba oil.

Materials
Colchicine was procured from Sigma-Aldrich Co.

Preparation of Colchicine Loaded Noisome
A method described previously by Shehata et al. was followed to formulate niosomal preparation using thin film hydration method [36]. Accordingly, non-ionic surfactant (span 60) and cholesterol with a molar ratio (1:1) were mixed together, followed by adding sufficient quantity of DSPE-PEG 2000 and then dissolved in chloroform using a round bottom flask. Afterward, under reduced pressure, the mixture was dried at 60 °C

Conclusions
Colchicine was successfully involved in niosomal preparation that is regarded as a promising drug delivery technique. For better application, the niosomal formulation was incorporated into various emulgel dosage forms. Box Behnken design approach was executed and came up with several niosomal emulgel formulas. The optimized of formula was selected based on values of certain investigated factors that was (1.04 g) jojoba oil, (0.9 g) Tween 80, and (0.803 g) Na alginate, and their obtained responses. The optimized formulation exhibited suitable physical properties including pH 6.73, viscosity 4598 cP, and spreadability 38.3 mm, in addition to an effective in vitro drug release study that reached 52.4% over 6 h. The developed formulations could mitigate hind paw inflammation induced by carrageenan since niosomal emulgel formulation containing jojoba oil could reduce paw swelling to reach 22.8%. In essence, the study revealed that the pharmacological action of colchicine was synergistically enhanced upon merging colchicine niosome with emulgel prepared with jojoba oil.

Materials
Colchicine was procured from Sigma-Aldrich Co.

Preparation of Colchicine Loaded Noisome
A method described previously by Shehata et al. was followed to formulate niosomal preparation using thin film hydration method [36]. Accordingly, non-ionic surfactant (span 60) and cholesterol with a molar ratio (1:1) were mixed together, followed by adding sufficient quantity of DSPE-PEG 2000 and then dissolved in chloroform using a round bottom flask. Afterward, under reduced pressure, the mixture was dried at 60 • C using rotary evaporator (Cole-Parmer, T-1602-21, Tokyo, Japan). A dried lipid film was formed on the wall of the flask, which was subjected to hydration using 5 mL dist. water containing colchicine (25 mg), while keeping agitation in water bath at 60 • C for 1 h to get niosomal solution. Consequently, the attained niosome was exposed to probe sonicator (XL-2000, Qsonica, Newtown, CT, USA) for 30 s to obtain the anticipated particle size.

Entrapment Efficiency
The entrapment efficiency of the developed colchicine-loaded niosome was considered as the percentage of colchicine carried by the preparations. Entrapment efficiency was assessed by employing centrifugation method. Niosomal formulation was added into Amicon ® ultra-4 (Ultracel-10K) and adjusted to be centrifuged at 6000 rpm using centrifuge (Andreas Hettich GmbH, Co.KG, Tuttlingen, Germany) while keeping the temperature at 4 • C for 1 h. The concentration of the free un-entrapped drug in the filtrate was measured spectrophotometrically at 350 nm (U.V. Spectrophotometer, JENWAY 6305) [45]. The entrapment efficiency of the drug was calculated by subtracting the amount of un-entrapped drug from the theoretical total drug added that was calculated using the following equation: where AF is the amount of the free un-entrapped colchicine and AT is the total amount of colchicine added.

Vesicular Size and Size Distribution
Size and polydispersity (PDI) of colchicine-loaded niosome vesicle were evaluated by measuring its dynamic light scattering using a Zetasizer apparatus (Malvern Instruments Ltd., Worcestershire, UK). All the experiments were performed in triplicate [46].

Stability Study
Investigating the stability of colchicine-loaded niosome was executed as per the guiding principal of International Conference on Harmonization (ICH). The study was implemented at two different conditions; 4 ± 1 • C and 25 ± 1 • C with humidity 60% for 1 and 3 months in terms of certain parameters, namely; the entrapment efficiency and vesicular size [47].

Preparation of Colchicine Niosomal Emulgel
As stated formerly, for the formulations to be applied properly over the skin, spread simply, and not easily detached from the treated area, it should demonstrate appropriate viscosity. In view of that, the developed niosomal formulation was incorporated into jojoba oil-based emulgel to attain colchicine niosomal emulgel. Primarily, hydrogel base was fabricated by sprinkling specified amount of Na alginate over 10 mL distilled water and mixed well by a magnetic stirrer (Jeio Tech TM-14SB; Medline Scientific, Oxfordshire, UK) until getting a uniform gel base. Next, emulsion was developed by mixing a definite amount of tween 80 with the distilled water for 10 min to form the aqueous phase. The aqueous phase was steadily added to the oily phase (jojoba oil) and the mixture vortexed well for 10 min using classic advanced vortex mixer (VELP Scintifica, Usmate Velate, Italy) until obtaining a consistent homogeneous emulsion. Eventually, emulgel formulation was obtained by mixing both, the hydrogel base with the developed emulsion using a mixer (Heidolph RZR1; Heidolph Instruments, Schwabach, Germany) for 10 min [48]. Table 1 displayed the quantified amount of different independent variables that were used for developing the niosomal formulations along with the values of the observed responses. In order to validate the influence of emulsion and predominantly jojoba oil in further studies, a niosomal gel containing colchicine was fabricated. In which, Na Alginate was added to 20 mL distilled water and stirred well till obtaining a gel, then mixed with the niosomal formulation (5 mL) to get colchicine niosomal gel.

Experimental Design Using BBD
In order to obtain an optimized niosomal emulgel formulation, a matrix of 17 formulas was constructed and fabricated using RSM. BBD is one of the most adequate techniques for analyzing the data and optimizing the formula; therefore, it was employed via constructing three factors, three levels (3 3 ) factorial design using Design-Expert version 12.0 software (Stat-Ease, Minneapolis, MN, USA). The factors representing the independent variables were selected as X 1 , X 2 , and X 3 related to oil concentration, surfactant concentration, and gelling agent concentration, respectively. The influence of the previous factors was studied on the responses R 1 (viscosity) and R 2 (in vitro release) of the developed niosomal emulgel. As apparent in Table 6, three different levels (−1, 0, 1) related to lowest, central, and the highest values of examined factors were established alongside their level of variations R 1 and R 2 corresponding to the responses. Statistical analysis of the data should be detected via analysis of variance (ANOVA) test. Moreover, correlating the obtained data from the statistical analysis is verified by definite model graphs such as 2D contour and 3D-response surface plots. In addition to a mathematical polynomial equation that could illustrate the response as follows: where R represents the detected response, b 0 represents the intercept; b 1 , b 2 , b 3 , b 12 , b 13 b 23 , b 11 , b 22 , and b 33 are the regression coefficients. X 1 , X 2 , and X 3 signify the main factors; X 1 X 2 , X 1 X 3 , and X 2 X 3 indicate the interactions between main factors and X 12 , X 22 , and X 32 point to the polynomial terms. Table 6. BBD data displaying independent variables with their level of variation and the examined dependent variables. Visual inspection of the tested formulations was carried out to examine the physical properties such as color and homogeneity.

Measurement of pH
Formulation's pH value is very important to ensure safety of the preparation and to avoid any probable skin irritation. Measuring the pH was accomplished using a standardized pH meter (MW802, Milwaukee Instruments, Szeged, Hungary).

Spreadability
This investigation is important for detecting the ability of the formulation to be spread evenly over the affected area by determining the spreadability diameter. In brief, a definite amount of the niosomal gel or emulgel formulation was added in between the two slides. About 0.5 g load was put for 1 min above the slides and the diameter of the spreading area was measured [50].

Assessment of In Vitro Drug Release Studies
The in vitro release investigation of drug from niosomal gel and emulgel preparations was inspected using Franz diffusion system (Logan Instruments Corp., FDC-6, Somerset, NJ, USA). Basically, quantified amounts of each formulation were placed separately over a cellophane membrane (MWCO 2000-15,000) (the donor) that covered the disillusion cell of the apparatus. The system was operated in 7 mL phosphate buffer (the acceptor), pH 7.4 adjusted at 37 ± 0.5 • C and rotated at 50 rpm. Samples were estimated spectrophotometrically at assorted time intervals (0.25, 0.5, 1, 2, 3, 4, 5, and 6 h) at λ max 350 nm. Each experiment was carried out in triplicate.

Release Kinetic Mechanism
Different kinetic models were applied in order to describe the mechanism by which the drug is released from the developed colchicine niosomal gel and emulgel in addition to the correlation coefficient (r2). Plotting drug concentration (C) versus time (t) would provide a profile of the in vitro study. If the model showed a linear plot and displayed the greatest value of (r2), the model is regarded as the best fitting. This study was conducted using the following models [51]: a. Zero order kinetic C = C0 + kt; b. First order kinetic C = C0 × ekt; c. Higuchi kinetic C = k × t 0.5 ; d. Korsmeyer-Peppas kinetic C = k × tn; where (C) represents the amount of colchicine released in time (t). (C0) indicates the value of (C) at time zero; (k) is the release constant; and (n) indicates the permeation rate exponent [52].

Animal
The animal breeding center at College of Science, King Faisal University provided us with Male Wister rats of about (220 to 250 g). The animals were handled by following the guidelines and ethics approved by Research Ethics Committee (REC) at King Faisal University (KFU-REC/2021-04-17). All animals were maintained in light and dark cycles, accommodated in an organized environment with an ambient temperature (25 ± 2 • C).

Carrageenan-Induced Rat Paw Edema Test
The action of colchicine as anti-inflammatory drug loaded into different formulations was evaluated via carrageenan-induced rat hind paw edema method. About 0.1 mL of 1% carrageenan in saline was injected subcutaneously into the left hind paw for edema induction, half an hour earlier to drug application [52]. Thirty six male Wister rats were allocated to six groups, each containing five rats of well-adjusted weight, and all groups were induced with inflammation as follows: